CN118103399A

CN118103399A - Pharmaceutical combinations of quinoline derivatives and anti-CD 47 antibodies

Info

Publication number: CN118103399A
Application number: CN202280062492.XA
Authority: CN
Inventors: 杨安琪; 张喜全; 于鼎; 吕鹏; 王训强; 田心; 陈琴
Original assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd; Nanjing Shunxin Pharmaceutical Co Ltd
Current assignee: Chia Tai Tianqing Pharmaceutical Group Co Ltd; Nanjing Shunxin Pharmaceutical Co Ltd
Priority date: 2021-09-30
Filing date: 2022-09-29
Publication date: 2024-05-28
Also published as: WO2023051669A1

Abstract

A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an anti-CD 47 antibody and a tyrosine kinase inhibitor. Also provided is the use of an anti-CD 47 antibody and a tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of cancer in a subject. Pharmaceutical combinations and kits comprising an anti-CD 47 antibody and a tyrosine kinase inhibitor are also provided. Wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof.

Description

Pharmaceutical combinations of quinoline derivatives and anti-CD 47 antibodies

Technical Field

The invention belongs to the field of biological medicine, and relates to application and pharmaceutical combination of quinoline derivatives and anti-CD 47 antibodies.

Background

Tyrosine kinases are a group of enzymes that catalyze the phosphorylation of protein tyrosine residues, play an important role in intracellular signal transduction, and are involved in the regulation, signaling and development of normal cells, and are also closely related to proliferation, differentiation, migration and apoptosis of tumor cells. Many receptor tyrosine kinases are involved in tumor formation and can be classified into Epidermal Growth Factor Receptor (EGFR), platelet Derived Growth Factor Receptor (PDGFR), vascular Endothelial Growth Factor Receptor (VEGFR), fibroblast Growth Factor Receptor (FGFR) and the like according to the structure of their extracellular regions. An Luoti Ni (Anlotinib) is a quinoline derivative tyrosine kinase inhibitor which plays a role in influencing tumor angiogenesis and proliferation signaling as a multi-target Tyrosine Kinase Inhibitor (TKI). Document WO2008112407 discloses in example 24 a tyrosine kinase inhibitor of the quinoline derivative class 1- [ [ [4- (4-fluoro-2-methyl-1H-indol-5-yl) oxy-6-methoxyquinolin-7-yl ] oxy ] methyl ] cyclopropylamine and a process for its preparation.

CD47 is a transmembrane glycoprotein widely expressed between multiple species and various tissues, also known as integrin-associated protein, a member of the immunoglobulin superfamily. Inhibitory receptor signaling protein α (sirpa) is one of the ligands for CD47, with CD47 binding to the NH2 terminal IgV-like domain of sirpa. Sirpa is expressed primarily in myeloid-derived cells, including macrophages, granulocytes, DC cells, mast cells, and their precursors, including hematopoietic stem cells. CD47 expression and/or activity has been implicated in a number of diseases and disorders. Several different studies have shown that almost all tumor cells and tumor tissues highly express CD47. The CD47 with high expression on the surface of the tumor cells releases signals of ' do ' T-me ' through combining with SIRPalpha on the surface of the macrophage, so that the macrophage in a tumor tissue infiltration area is not only in harmony with the tumor cells, but also can play a role in inhibiting effector T cells by promoting proliferation of blood vessels in the tumor, and promote expansion and growth of the tumor cells. Drugs such as anti-CD 47 antibodies against CD 47-sirpa axis are currently entering clinical trials.

There is still an unmet clinical need for treatment of cancer.

Disclosure of Invention

The present invention provides the use of an anti-CD 47 antibody and a tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of cancer in a subject, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof.

The invention provides a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an anti-CD 47 antibody and a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical combination comprising: (a) An anti-CD 47 antibody, and (b) a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical combination for treating cancer comprising: (a) An anti-CD 47 antibody, and (b) a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof.

The invention also provides a kit comprising the pharmaceutical combination.

Drawings

FIG. 1 is a graph showing the effect of anti-CD 47 antibodies on survival of mice.

Figure 2 is a graph showing the change in body weight of mice over time after a single administration of an anti-CD 47 antibody.

FIG. 3 shows the rate of change of body weight of mice at various times after a single administration of anti-CD 47 antibody.

Figure 4 is a graph showing RBC changes over time in mice after a single administration of anti-CD 47 antibody.

Figure 5 shows the rate of change of RBC in mice at various times after a single administration of anti-CD 47 antibody.

FIG. 6 is a graph showing the HGB content of mice after single administration of an anti-CD 47 antibody over time.

FIG. 7 shows the rate of change of mouse HGB at various times after a single administration of an anti-CD 47 antibody.

Fig. 8 shows the change in tumor volume of human colorectal carcinoma divi mice subcutaneous grafts after anti-CD 47 antibodies, an Luoti nim alone or in combination.

FIG. 9 shows the change in body weight of tumor-bearing mice after anti-CD 47 antibodies, an Luoti Ni hydrochloride alone or in combination.

Detailed Description

Terminology

The term "antibody" is used in its broadest sense and covers a variety of antibody structures, including natural and artificial antibodies of various structures, including but not limited to monoclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), single chain antibodies, antibody fragments, and the like, so long as they exhibit the desired antigen-binding activity. The antibody may be an intact antibody. An antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') 2, single chain antibody molecules (e.g., scFv), and single domain antibodies.

The term "isotype" refers to the type of antibody encoded by the heavy chain constant region gene.

The term "chimeric antibody" is an antibody as follows: the antibodies have at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from a species; and at least a portion of a constant region derived from another species. For example, in some embodiments, a chimeric antibody may comprise murine variable regions and human constant regions.

"Humanized antibodies" are the following antibodies: the antibodies contain Complementarity Determining Regions (CDRs) derived from a non-human antibody; and framework and constant regions derived from human antibodies. For example, a humanized antibody that binds CD47 provided herein can comprise CDRs derived from one or more murine antibodies as well as human framework and constant regions.

The term "variable domain" or "variable region" refers to a domain of an antibody that relates to the binding of the antibody to an antigen. For example, natural four-chain antibodies (e.g., derived from humans, mice, etc.) have a heavy chain variable region (VH) and a light chain variable region (VL), and heavy chain-only antibodies derived from animals such as camelidae or sharks have a single variable domain. In most cases, each variable domain of a natural antibody consists essentially of four "framework regions" and three "complementarity determining regions". The four frame regions are referred to as frame region 1 (FR 1), frame region 2 (FR 2), frame region 3 (FR 3), and frame region 4 (FR 4), respectively; the framework regions are separated by three Complementarity Determining Regions (CDRs) known in the art and hereinafter as complementarity determining region 1 (CDR 1), complementarity determining region 2 (CDR 2), and complementarity determining region 3 (CDR 3), respectively. Thus, the general structure of the variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. As a specific example, the general structure of the heavy chain variable region can be expressed as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. As a specific example, the general structure of the light chain variable region can be expressed as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. The variable domains confer specificity to the antigen to the antibody by having an antigen binding site.

"CDR" (complementarity determining region), also known as "hypervariable region" (HVR), generally refers to each region of an antibody variable region that is highly variable in sequence and/or forms a structurally defined loop. Natural four-chain antibodies typically comprise six CDRs, three in the heavy chain variable region, heavy chain CDR1 (HCDR 1), heavy chain CDR2 (HCDR 2), and heavy chain CDR3 (HCDR 3), respectively, and three in the light chain variable region, light chain CDR1 (LCDR 1), light chain CDR2 (LCDR 2), and light chain CDR3 (LCDR 3), respectively. Heavy chain-only antibodies or single variable domains typically have three CDRs (CDR 1, CDR2, and CDR 3).

There are many methods currently available for partitioning defined CDRs. Wherein the Kabat definition divides CDRs based on sequence variability and is most commonly used (Elvin a. Kabat, et al, sequences of Proteins of Immunological Interest, 5th edition, public HEALTH SERVICE, national Institutes of Health, bethesda, md. (1991)); the Chothia definition is based on the position (Cyrus Chothia,et al,Canonical Structures for the Hypervariable Regions of Immunoglobulins,J.Mol.Biol.196:901-917(1987)).AbM of the structural loop, which is a compromise between the Kabat definition and Chothia definition, and is used by Oxford Molecular AbM antibody modeling software. The basis for the definition of "contact" is an analysis of the crystal structure of the available complexes. It should be noted, however, that the boundaries of CDRs of the same antibody variable region obtained based on different method division definitions may differ, i.e., CDR sequences of the same antibody variable region defined by different method division definitions may differ. Thus, where specific CDR sequences defined by certain divisions of the invention are involved in defining antibodies, the scope of the antibodies also encompasses antibodies that are converted to CDR sequence definitions of other arbitrary definitions (e.g., chothia, abM definitions, etc.).

The term "framework region" (FR) is an amino acid residue of those variable domains other than the CDR residues defined herein.

The term "isolated" refers to a compound of interest (e.g., an antibody or nucleic acid) that has been isolated from its natural environment.

As used herein, the term "EC50" refers to the effective concentration, 50% of the maximum response of an antibody. As used herein, the term "IC50" refers to the inhibitory concentration, 50% of the maximum response of an antibody. Both EC50 and IC50 may be measured by ELISA or FACS analysis or any other method known in the art.

The term "K _D" as used herein refers to the equilibrium dissociation constant, expressed as molar concentration (M). The K _D value of an antibody can be determined using methods well known in the art. One preferred method of determining antibody K _D is to use surface plasmon resonance (surface plasmon resonance), more preferably using a biosensor system, such as the Biacore system.

As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, anchors, horses, cows, chickens, amphibians, reptiles, and the like. Preferably, the subject according to the invention is a human. Unless indicated, the terms "patient" or "subject" are used interchangeably.

As used herein, "cancer" refers to a physiological condition in a mammal that is characterized by unregulated cell growth in general. Uncontrolled cell division and growth results in the formation of malignant tumors that invade adjacent tissues, and may also metastasize to distal parts of the body through the lymphatic system or blood flow. "cancer" or "cancer tissue" may include tumors.

"Recurrent" cancer is cancer that regenerates at an initial site or a distant site after responding to an initial treatment (e.g., surgery). A "locally recurrent" cancer is a cancer that occurs at the same location after treatment as the previously treated cancer.

"Unresectable" cancers are not removed by surgery.

"Metastatic" cancer refers to cancer that spreads from one part of the body (e.g., lung, stomach, etc.) to another part of the body.

The term "treatment" refers to attempting to alter the natural course of a disease in a treated individual and may be for the purpose of preventing or clinical intervention performed during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis.

As used herein, the term "therapeutically effective amount" refers to the amount of a compound, composition, or pharmaceutical combination necessary to provide a therapeutic benefit to a subject.

The term "pharmaceutically acceptable" is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salts" includes salts of a base ion with a free acid or salts of a acid ion with a free base, including for example, hydrochloride, hydrobromide, nitrate, sulfate, phosphate, formate, acetate, trifluoroacetate, fumarate, oxalate, maleate, citrate, succinate, methanesulfonate, benzenesulfonate or p-toluenesulfonate salts, preferably hydrochloride, hydrobromide, sulfate, formate, acetate, trifluoroacetate, fumarate, maleate, methanesulfonate, p-toluenesulfonate, sodium, potassium, ammonium, amino acid salts and the like.

The term "non-fixed combination" refers to the simultaneous, concurrent or sequential administration of two or more active ingredients as separate entities (e.g., pharmaceutical compositions, formulations) to a subject, wherein the administration of the active ingredients to the subject achieves a therapeutically effective amount level, without specific time limitations. An example of a non-fixed combination is a cocktail therapy, e.g., administration of 3 or more active ingredients. In a non-fixed combination, the individual active ingredients may be packaged, marketed or administered as a fully independent pharmaceutical composition.

The term "fixed dose" refers to a dose administered to a patient irrespective of the patient's body weight or Body Surface Area (BSA). A fixed dose is therefore specified as an absolute amount of agent (e.g., anti-CD 47 antibody) rather than as, for example, a mg/kg dose; for example, 80kg of humans and 100kg of humans will receive the same dose of antibody (e.g., 800mg of anti-CD 47 antibody). The administration of the dose may also be calculated according to the weight or body surface area of the patient, as opposed to a fixed dose, for example, according to the weight of the patient at a dose of 0.5mg/kg to 70 mg/kg.

The term "single dose" refers to the smallest packaging unit containing a quantity of a drug, e.g., a box of seven capsules, each capsule being a single dose; or a single dose per bottle of injectate. The terms "single dose" and "unit dose" have the same meaning and are used interchangeably herein.

"Pharmaceutical composition" refers to a composition comprising an active ingredient and a pharmaceutically acceptable carrier.

"Percent (%) identity" of amino acid sequences refers to the percentage of amino acid residues in an aligned sequence that are identical to the amino acid residues of a particular amino acid sequence shown herein when gaps are introduced in the aligned sequence to the particular amino acid sequence shown herein and if necessary to achieve the maximum percent sequence identity, and no conservative substitutions are made as part of the sequence identity. The amino acid sequence identity alignment can be performed in a variety of ways within the skill in the art, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm needed to obtain the maximum alignment over the entire length of the compared sequences.

The terms "Xn" and "Xaa" are equivalent and refer to an unspecified amino acid (Unspecified Amino Acid), the scope of which is specified by the subsequent definition in the relevant expression.

The terms "comprises," "comprising," or "includes" and variations thereof are to be interpreted as referring to "including but not limited to," means that the elements, components, and steps recited are included in addition to other non-recited elements, components, and steps.

In this document, singular terms encompass plural referents and vice versa, unless the context clearly dictates otherwise.

All patents, patent applications, and other identified publications are expressly incorporated herein by reference for the purpose of description and disclosure. These publications are provided solely for their disclosure prior to the filing date of the present application. All statements as to the date or representation as to the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the correctness of the dates or contents of these documents. Moreover, any reference herein to such publications in any country is not an admission that such publications are part of the common general knowledge in the art. Various aspects of the application will be described in further detail in the following sections.

Anti-CD 47 antibodies are suitable for use in combination with the tyrosine kinase inhibitors to treat cancer. Furthermore, the combination may have one or more of the following advantages:

(1) In some experiments, it was found that anti-CD 47 antibodies in combination with the tyrosine kinase inhibitors may enhance the anti-tumor effect of the single agents, e.g. killing tumor cells, inhibiting tumor growth or/and eliminating tumors, etc. In particular, it has good therapeutic effect or/and safety, for example, in some solid tumors such as colorectal cancer.

(2) Anti-CD 47 antibodies and the tyrosine kinase inhibitors may provide a treatment that is well tolerated in patients, with good safety, e.g., with fewer adverse reactions or/and complications.

(3) The anti-CD 47 antibodies and the tyrosine kinase inhibitors may allow for administration of smaller amounts (e.g., administered doses or/and total administered amounts) and benefit as compared to single or other certain known drugs or combinations.

In some embodiments, the pharmaceutically acceptable salt of the compound of formula I is the hydrochloride, preferably the dihydrochloride, of 1- [ [ [4- (4-fluoro-2-methyl-1H-indol-5-yl) oxy-6-methoxyquinolin-7-yl ] oxy ] methyl ] cyclopropylamine.

In some embodiments, the cancer is a solid tumor. In some embodiments, the solid tumor is an advanced solid tumor.

In some embodiments, the cancer is primary, recurrent, or metastatic. In some specific embodiments, the cancer is a recurrent or/and metastatic solid tumor. In some more specific embodiments, the cancer is a recurrent or/and metastatic advanced solid tumor.

In some embodiments, the solid tumor is colorectal cancer.

In some embodiments, the colorectal cancer is primary colorectal cancer and/or secondary colorectal cancer.

In some embodiments, the colorectal cancer is colorectal cancer that failed prior treatment, e.g., the colorectal cancer is colorectal cancer that failed radiation and/or chemotherapy and/or targeted drug therapy.

In some embodiments, the colorectal cancer is colorectal adenocarcinoma, in some embodiments, the colorectal adenocarcinoma is sieve-like acne adenocarcinoma, medullary carcinoma, micro-papillary carcinoma, mucous adenocarcinoma, serrated adenocarcinoma, or print-stop cell carcinoma.

In some embodiments, the colorectal cancer is RAS wild-type colorectal cancer; in some embodiments, the colorectal cancer is RAS wild-type colorectal cancer that fails to be treated with radiation and/or chemotherapy and/or targeted drugs. In some embodiments, the colorectal cancer is BRAF wild-type colorectal cancer; in some embodiments, the colorectal cancer is BRAF wild-type colorectal cancer that fails radiotherapy and/or chemotherapy and/or targeted drug therapy. In some embodiments, the colorectal cancer is colorectal cancer of RAS and/or BRAF wild-type. In some embodiments, the colorectal cancer is PIK3CA wild-type colorectal cancer; in some embodiments, the colorectal cancer is PIK3CA wild-type colorectal cancer that fails radiotherapy and/or chemotherapy and/or targeted drug therapy.

In some embodiments, the colorectal cancer is a RAS mutant colorectal cancer; in some embodiments, the colorectal cancer is a RAS mutant colorectal cancer that fails to be treated with radiation and/or chemotherapy and/or targeted drugs. In some embodiments, the colorectal cancer is BRAF mutant colorectal cancer; in some embodiments, the colorectal cancer is a BRAF mutant colorectal cancer that fails to be treated with radiation and/or chemotherapy and/or targeted drugs. In some embodiments, the colorectal cancer is a RAS/BRAF mutant colorectal cancer. In some embodiments, the colorectal cancer is PIK3CA mutant colorectal cancer; in some embodiments, the colorectal cancer is a PIK3CA mutant colorectal cancer that fails to be treated with radiation and/or chemotherapy and/or targeted drugs. In some typical embodiments, the colorectal cancer is advanced and/or metastatic colorectal cancer with RAS mutations; in some typical embodiments, the colorectal cancer is advanced and/or metastatic colorectal cancer of KRAS mutations. In some typical embodiments, the colorectal cancer is advanced and/or metastatic colorectal cancer with failed radiotherapy and/or chemotherapy and/or targeted drug therapy of RAS mutations. In some typical embodiments, the colorectal cancer is advanced and/or metastatic colorectal cancer with KRAS mutations that failed radiotherapy and/or chemotherapy and/or targeted drug therapy. In some typical embodiments, the colorectal adenocarcinoma is advanced and/or metastatic colorectal adenocarcinoma with RAS mutations.

In some embodiments, the RAS mutations include, but are not limited to, KRAS and NRAS mutations. In some embodiments, the RAS mutation is a KRAS mutation. In some embodiments, the RAS mutation is an NRAS mutation. In some embodiments, the colorectal cancer is advanced and/or metastatic colorectal cancer with a camptothecins, platinum, fluoropyrimidine derivatives, and/or EGFR inhibitors that failed treatment with KRAS mutations.

In some embodiments, the colorectal cancer is pMMR (mismatch repair function complete) or/and MSS (microsatellite stabilized) colorectal cancer. In some embodiments, the colorectal cancer is dMMR (mismatch repair function defect) or/and MSI (microsatellite instability) colorectal cancer.

In some embodiments, a therapeutically effective amount of an anti-CD 47 antibody and the tyrosine kinase inhibitor are administered to a subject having cancer. In some embodiments, the therapeutic dose of the anti-CD 47 antibody is administered at the patient's weight in the range of 0.5mg/kg to 70mg/kg, for example 0.5mg/kg to 40mg/kg,15mg/kg to 30mg/kg,25mg/kg to 35mg/kg, or 15mg/kg to 35mg/kg. Suitable dosages include, for example 1mg/kg、2mg/kg、3mg/kg、4mg/kg、5mg/kg、6mg/kg、7mg/kg、8mg/kg、9mg/kg、10mg/kg、15mg/kg、16mg/kg、17mg/kg、18mg/kg、19mg/kg、20mg/kg、21mg/kg、22mg/kg、23mg/kg、24mg/kg、25mg/kg、26mg/kg、27mg/kg、28mg/kg、29mg/kg、30mg/kg、31mg/kg、32mg/kg、33mg/kg、34mg/kg、35mg/kg、36mg/kg、37mg/kg、38mg/kg、39mg/kg、40mg/kg、41mg/kg、42mg/kg、43mg/kg、44mg/kg、45mg/kg、50mg/kg、55mg/kg、60mg/kg、65mg/kg or 70mg/kg.

In some embodiments, a therapeutic dose of anti-CD 47 antibody may also be administered to a subject having cancer at a fixed dose, in some embodiments, the fixed dose of anti-CD 47 antibody is 60mg to 3000mg, such as 60mg to 2000mg,600mg to 1800mg, or 1000mg to 1800mg, suitable fixed doses including, for example, 60mg、100mg、180mg、360mg、500mg、600mg、700mg、800mg、900mg、1000mg、1100mg、1200mg、1300mg、1400mg、1500mg、1600mg、1700mg、 1800mg、1900mg、2000mg、2500mg or 3000mg.

In some embodiments, the anti-CD 47 antibody is administered at a therapeutic dose once, twice or more every 3-21 days, e.g., every 3-14 days, or 7-14 days. A suitable dosing frequency is, for example, twice weekly, once weekly (q 1 w), once every 2 weeks (q 2 w), or once every 3 weeks (q 3 w)) a therapeutic dose of an anti-CD 47 antibody is administered.

In some embodiments, the anti-CD 47 antibody may be administered at a therapeutic dose typically between 1 to 6 times (e.g., 1,2, 3,4, 5, or 6 times) to treat cancer (such as liver cancer), but 7,8,9, 10, 11, 12, 13, 14, or more therapeutic doses may be administered.

In some embodiments, a therapeutic dose of anti-CD 47 antibody is administered to a subject having cancer for a treatment period of every 2-6 weeks, e.g., every 2 weeks (14 days), every 3 weeks (21 days), every 4 weeks (28 days), every 5 weeks (35 days), or every 6 weeks (42 days).

In some embodiments, administration of an anti-CD 47 antibody by escalation of the dose is allowed to achieve the therapeutic dose.

In some embodiments, a sub-therapeutic dose of an anti-CD 47 antibody is allowed to be administered below the therapeutic dose prior to administration of the therapeutic dose of the anti-CD 47 antibody.

In some embodiments, the tyrosine kinase inhibitor is administered at a dose of 6mg to 20mg, for example 6mg to 16mg,6mg to 12mg, or 8mg to 12mg. Suitable administration doses of the tyrosine kinase inhibitor may be, for example, 6mg, 8mg, 10mg, 12mg, 14mg, 16mg, 18mg or 20mg.

In some embodiments, the tyrosine kinase inhibitor may be administered one or more times daily, preferably once.

In some embodiments, the tyrosine kinase inhibitor is administered once daily for a 2 week regimen of 2 weeks off. In some embodiments, the tyrosine kinase inhibitor is administered once daily for 2 weeks with 1 week of discontinuation. In certain specific embodiments, the tyrosine kinase inhibitor is administered orally at a dose of 8mg, 10mg or 12mg once daily for 2 weeks, followed by 1 week of administration.

In some embodiments, the anti-CD 47 antibody and the tyrosine kinase inhibitor are administered simultaneously, sequentially or at intervals.

The present invention also provides a pharmaceutical combination comprising:

(a) anti-CD 47 antibodies, and

(B) A tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt of the compound of formula I is the hydrochloride, preferably the dihydrochloride, of 1- [ [ [4- (4-fluoro-2-methyl-1H-indol-5-yl) oxy-6-methoxyquinolin-7-yl ] oxy ] methyl ] cyclopropylamine.

In some embodiments, the pharmaceutical combination is a non-fixed combination. The anti-CD 47 antibody and the tyrosine kinase inhibitor in the non-fixed combination are each in the form of a pharmaceutical composition.

In some embodiments, the tyrosine kinase inhibitor is present in a pharmaceutical composition having at least one single dose, wherein the single dose is 6mg to 20mg, e.g., 6mg to 16mg, 6mg to 12mg, or 8mg to 12mg. Suitable single doses of the tyrosine kinase inhibitor include, for example, 6mg, 8mg, 10mg, 12mg, 14mg, 16mg, 18mg or 20mg.

In some embodiments, the anti-CD 47 antibody is in a pharmaceutical composition suitable for administration in a fixed dose.

In some embodiments, the invention provides a kit comprising the pharmaceutical combination. The kit may include instructions for administering the pharmaceutical combination to a subject having cancer. The kit may also include other materials as required from a commercial and user standpoint, for example, may include other buffers, diluents, needles, syringes, and the like.

In some embodiments, the pharmaceutical combination is for use in treating cancer described herein above.

In some embodiments, there is provided a pharmaceutical combination for treating the cancers described herein above comprising:

(a) anti-CD 47 antibodies, and

In the pharmaceutical combination, the anti-CD 47 antibody and the tyrosine kinase inhibitor may be administered by the routes, the doses, the time/interval of administration, etc. referred to in the above uses or methods, and will not be described herein.

A compound of formula I or a pharmaceutically acceptable salt thereof

As used herein, the chemical name of the compound of formula I is 1- [ [ [4- (4-fluoro-2-methyl-1H-indol-5-yl) oxy-6-methoxyquinolin-7-yl ] oxy ] methyl ] cyclopropylamine, having the structural formula:

In the present application, all references to An Luoti are to compounds of formula I. An Luoti Ni hydrochloride is the hydrochloride salt of the compound of formula I.

The compounds of formula I may be administered in its free base form, or in the form of salts, hydrates and prodrugs thereof, which are converted in vivo to the free base form of the compounds of formula I. For example, pharmaceutically acceptable salts of the compounds of formula I are within the scope of the application, which salts may be produced from different organic and inorganic acids according to methods well known in the art.

In some embodiments, the compound of formula I is administered as the hydrochloride salt. In some embodiments, the compound of formula I is administered as the monohydrochloride salt. In some embodiments, the compound of formula I is administered as the dihydrochloride salt. In some embodiments, the compound of formula I is administered in crystalline form as the hydrochloride salt. In certain embodiments, the compound of formula I is administered in crystalline form as the dihydrochloride salt of the compound.

The compound of formula I, or a pharmaceutically acceptable salt thereof, may be administered by a variety of routes including, but not limited to, those selected from the group consisting of: oral, parenteral, intraperitoneal, intravenous, intraarterial, transdermal, sublingual, intramuscular, rectal, buccal, intranasal, inhalation, vaginal, intraocular, topical, subcutaneous, intrafat, intra-articular, intraperitoneal and intrathecal. In a particular embodiment, the administration is by oral administration.

Herein, unless otherwise indicated, the dosages and ranges provided for them are based on the molecular weight of the free base form of the compound of formula I.

Anti-CD 47 antibodies

Some exemplary anti-CD 47 antibodies suitable for use in the above uses, methods or pharmaceutical combinations are provided below.

In certain embodiments, in addition to binding to human CD47 or/and cynomolgus monkey CD47, provided anti-CD 47 antibodies also exhibit a number of other desirable features for targeted therapy. In particular, these desirable features may be at least one of, for example, a K _D value of 1.53E-08 or less binding to CD47, blocking CD47 binding to sirpa, promoting macrophage-mediated phagocytosis of CD47 expressing cells, not significantly inducing apoptosis of CD4 ⁺ T cells, not causing substantial erythropenia, anemia or erythrocyte agglutination, the melting temperature TT62 ℃, polymerization temperature TaggT ℃, etc. of the antigen binding polypeptide or antigen binding portion thereof. In some embodiments, the anti-CD 47 antibody exhibits a better therapeutic effect; in some embodiments, the anti-CD 47 antibodies exhibit reduced side effects.

In some embodiments, the anti-CD 47 antibody is chimeric, humanized or human. In a particular embodiment, the anti-CD 47 antibody is humanized.

In some embodiments, the anti-CD 47 antibody is an IgG1, igG2, igG3, or IgG4 isotype. In some embodiments, the anti-CD 47 antibody is an IgG4 isotype. In some embodiments, the anti-CD 47 antibody is an IgG4 isotype and contains an S228P amino acid substitution according to EU numbering.

In some embodiments, the anti-CD 47 antibody comprises:

a heavy chain variable region comprising: comprising SEQ ID NO:1, comprising the amino acid sequence set forth in SEQ ID NO:6, and HCDR2 comprising the amino acid sequence of SEQ ID NO:11, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:16, comprising the amino acid sequence set forth in SEQ ID NO:21, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:26, and LCDR3 of the amino acid sequence shown in seq id no.

In some embodiments, the anti-CD 47 antibody comprises:

A heavy chain variable region comprising: comprising SEQ ID NO:2, HCDR1 comprising the amino acid sequence of SEQ ID NO:7, and HCDR2 comprising the amino acid sequence of SEQ ID NO:12, HCDR3 of an amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:17, LCDR1 comprising the amino acid sequence of SEQ ID NO:22, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:27, and LCDR3 of the amino acid sequence shown in seq id no.

In some embodiments, the anti-CD 47 antibody comprises:

A heavy chain variable region comprising: comprising SEQ ID NO:3, HCDR1 comprising the amino acid sequence of SEQ ID NO:8, and HCDR2 comprising the amino acid sequence of SEQ ID NO:13, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:18, comprising the amino acid sequence set forth in SEQ ID NO:23, and LCDR2 comprising the amino acid sequence of SEQ ID NO:28, and LCDR3 of the amino acid sequence shown in seq id no.

In some embodiments, the anti-CD 47 antibody comprises:

A heavy chain variable region comprising: comprising SEQ ID NO:4, HCDR1 comprising the amino acid sequence of SEQ ID NO:9, and an HCDR2 comprising the amino acid sequence of SEQ ID NO:14, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:19, comprising the amino acid sequence set forth in SEQ ID NO:24, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:32, and LCDR3 of the amino acid sequence shown in seq id no.

In some embodiments, the anti-CD 47 antibody comprises:

A heavy chain variable region comprising: comprising SEQ ID NO:5, an HCDR1 comprising the amino acid sequence of SEQ ID NO:10, and an HCDR2 comprising the amino acid sequence of SEQ ID NO:15, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:20, comprising the amino acid sequence set forth in SEQ ID NO:25, and an LCDR2 comprising an amino acid sequence of SEQ ID NO:30, and LCDR3 of the amino acid sequence shown in seq id no.

In some embodiments, the anti-CD 47 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33. 35, 37, 39, 41, 83, 85 or 87, and HCDR1, HCDR2 and HCDR3 of a variable region sequence of SEQ ID NO: 34. 36, 38, 40, 42, 84, 86 or 88, LCDR1, LCDR2 and LCDR3 of a variable region sequence as shown. In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:83, and HCDR1, HCDR2 and HCDR3 of a variable region sequence of an anti-CD 47 antibody comprising the amino acid sequence of SEQ ID NO:84, LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in seq id no. In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:85, and HCDR1, HCDR2 and HCDR3 of a variable region sequence of an anti-CD 47 antibody comprising the amino acid sequence of SEQ ID NO:86, LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in seq id no. In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:87, and HCDR1, HCDR2 and HCDR3 of a variable region sequence of an anti-CD 47 antibody comprising the light chain variable region sequence of SEQ ID NO:88, LCDR1, LCDR2 and LCDR3 of the variable region sequence shown in seq id no.

In some embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises a sequence that hybridizes to SEQ ID NO: 33. 35, 37, 39, 41, 83, 85, or 87, has an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, the light chain variable region of the anti-CD 47 antibody comprises a sequence that hybridizes to SEQ ID NO: 34. 36, 38, 40, 42, 84, 86, or 88 has an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical.

In some embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises a sequence that hybridizes to SEQ ID NO:39, and the light chain variable region of the anti-CD 47 antibody comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:40, has an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. Further, in some such embodiments, the anti-CD 47 antibody further comprises a polypeptide comprising SEQ ID NO:4, HCDR1 comprising the amino acid sequence of SEQ ID NO:9, HCDR2 comprising the amino acid sequence of SEQ ID NO:14, HCDR3 comprising the amino acid sequence of SEQ ID NO:19, LCDR1 comprising the amino acid sequence of SEQ ID NO:24 and an LCDR2 comprising the amino acid sequence of SEQ ID NO:32, and LCDR3 of the amino acid sequence shown in seq id no.

In some embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises a sequence that hybridizes to SEQ ID NO:41, and the light chain variable region of the anti-CD 47 antibody comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:42, has an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. Further, in some such embodiments, the anti-CD 47 antibody further comprises a polypeptide comprising SEQ ID NO:5, HCDR1 comprising the amino acid sequence of SEQ ID NO:10, HCDR2 comprising the amino acid sequence of SEQ ID NO:15, HCDR3 comprising the amino acid sequence of SEQ ID NO:20, LCDR1 comprising an amino acid sequence shown as SEQ ID NO:25 and an LCDR2 comprising the amino acid sequence of SEQ ID NO:30, and LCDR3 of the amino acid sequence shown in seq id no.

In some embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises a sequence that hybridizes to SEQ ID NO:83, and the light chain variable region of the anti-CD 47 antibody comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:84 has an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. Further, in some such embodiments, the anti-CD 47 antibody further comprises a polypeptide comprising SEQ ID NO:4, HCDR1 comprising the amino acid sequence of SEQ ID NO:9, HCDR2 comprising the amino acid sequence of SEQ ID NO:14, HCDR3 comprising the amino acid sequence of SEQ ID NO:19, LCDR1 comprising the amino acid sequence of SEQ ID NO:24 and an LCDR2 comprising the amino acid sequence of SEQ ID NO:32, and LCDR3 of the amino acid sequence shown in seq id no.

In some embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises a sequence that hybridizes to SEQ ID NO:87, and the light chain variable region of the anti-CD 47 antibody comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:88, has an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. Further, in some such embodiments, the anti-CD 47 antibody further comprises a polypeptide comprising SEQ ID NO:5, HCDR1 comprising the amino acid sequence of SEQ ID NO:10, HCDR2 comprising the amino acid sequence of SEQ ID NO:15, HCDR3 comprising the amino acid sequence of SEQ ID NO:20, LCDR1 comprising an amino acid sequence shown as SEQ ID NO:25 and an LCDR2 comprising the amino acid sequence of SEQ ID NO:30, and LCDR3 of the amino acid sequence shown in seq id no.

In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:39, and the light chain variable region of said anti-CD 47 antibody comprises the amino acid sequence set forth in SEQ ID NO:40, and a polypeptide having the amino acid sequence shown in seq id no.

In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:41, and the light chain variable region of said anti-CD 47 antibody comprises the amino acid sequence set forth in SEQ ID NO: 42.

In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:83, and the light chain variable region of said anti-CD 47 antibody comprises the amino acid sequence set forth in SEQ ID NO: 84.

In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:87, and the light chain variable region of said anti-CD 47 antibody comprises the amino acid sequence set forth in SEQ ID NO:88, and a sequence of amino acids shown in seq id no.

In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:81, and the light chain variable region of said anti-CD 47 antibody comprises the amino acid sequence set forth in SEQ ID NO: 82.

In some specific embodiments, the heavy chain variable region of the anti-CD 47 antibody comprises the amino acid sequence of SEQ ID NO:79, and the light chain variable region of said anti-CD 47 antibody comprises the amino acid sequence set forth in SEQ ID NO:80, and an amino acid sequence shown in seq id no.

In other specific embodiments, the heavy chain variable region and the light chain variable region of the anti-CD 47 antibody comprise an amino acid sequence selected from the group consisting of SEQ ID NO：33/34、35/36、37/38、43/44、45/46、47/48、49/50、51/52、53/54、55/56、57/58、59/60、61/62、63/64、65/66、67/68、69/70、71/72、73/74、75/76、77/78、85/86.

In some embodiments, the anti-CD 47 antibody comprises a heavy chain and a light chain. In some embodiments, the heavy chain of the anti-CD 47 antibody comprises a sequence that hybridizes to SEQ ID NO:181, and the light chain of the anti-CD 47 antibody comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:183 have an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical. In a specific embodiment, the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:181 and a heavy chain according to SEQ ID NO: 183. In other embodiments, the heavy chain of the anti-CD 47 antibody comprises a sequence that hybridizes to SEQ ID NO:185, and the light chain of the anti-CD 47 antibody comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:187, an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity. In a specific embodiment, the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:185 and a heavy chain according to SEQ ID NO: 187. In other embodiments, the anti-CD 47 antibody may further comprise the heavy and light chains of the Xi2H8、Xi16E5、Xi2B2、Xi3F10、Xi14A9、hz3F10-1.1、hz3F10-2.1、hz3F10-3.1、hz3F10-4.1、hz3F10-5.1、hz3F10-6.1、hz3F10-1.2、hz3F10-2.2、hz3F10-3.2、hz3F10-4.2、hz3F10-5.2、hz3F10-6.2、hz16E5-1.1、hz16E5-1.3、hz16E5-1.2、hz16E5-3.2、hz16E5-3.1、hz16E5-3.3 antibody.

Also provided are some exemplary monoclonal antibodies that bind CD47, including chimeric versions of the 2B2, 2H8, 3F10, 16E5, and 14A9 antibodies (Xi 2B2, xi2H8, xi3F10, xi16E5, and Xi14 A9) and humanized variants of 3F10, 16E5, 14 A9. The amino acid sequences of the HCDRs (HCDR 1, HCDR2, and HCDR 3) of the exemplary CD 47-binding antibodies provided herein are provided in table S1 below, the amino acid sequences of the LCDRs (LCDR 1, LCDR2, and LCDR 3) are provided in table S2 below, and the amino acid sequences of a portion of the antibody variable regions and a portion of the full-length heavy and light chains of the antibodies are provided in tables S3, S4 below.

Table S1: heavy chain CDR sequences of anti-CD 47 antibodies

Table S2: light chain CDR sequences of anti-CD 47 antibodies

Table S3: variable region sequences of partial anti-CD 47 antibodies

Table S4: sequences of full length heavy and light chains of partial anti-CD 47 antibodies

In certain specific embodiments, the anti-CD 47 antibody is hz14A9-2.3 or hz14A9-2.4.

In other embodiments, the anti-CD 47 antibody may be other known antibodies, such as magrolimab, letaplimab, AK117, AO-176, lemzoparlimab, and the like.

The anti-CD 47 antibodies may be administered by a variety of routes, in one embodiment, intravenously.

The anti-CD 47 antibodies may be prepared using methods well known in the art. Culturing a host cell comprising a nucleic acid encoding said anti-CD 47 antibody, e.g., under conditions suitable for expression of said anti-CD 47 antibody, and recovering said anti-CD 47 antibody from said host cell or host cell culture medium. To produce anti-CD 47 antibodies, the nucleic acid encoding the antibodies is isolated and inserted into one or more vectors for further cloning or/and expression in a host cell. The nucleic acid may be obtained by various methods known in the art, such as gene cloning, gene splicing, chemical synthesis, and the like. The recovery can be performed by centrifugation, affinity chromatography, or the like.

Other exemplary embodiments are provided herein, but are not limited to:

Embodiment 1. Use of an anti-CD 47 antibody and a tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of cancer in a subject, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof,

Embodiment 2. A method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of an anti-CD 47 antibody and a tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof,

Embodiment 3. A pharmaceutical combination comprising:

(a) anti-CD 47 antibodies, and

(B) A tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof,

Embodiment 4. The pharmaceutical combination, use or method according to any of embodiments 1 to 3, wherein the pharmaceutically acceptable salt of the compound of formula I is the hydrochloride, preferably the dihydrochloride, of 1- [ [ [4- (4-fluoro-2-methyl-1H-indol-5-yl) oxy-6-methoxyquinolin-7-yl ] oxy ] methyl ] cyclopropylamine.

Embodiment 5. The pharmaceutical combination, method or use according to any one of embodiments 1-4, wherein the anti-CD 47 antibody comprises:

(i) A heavy chain variable region comprising:

Comprising SEQ ID NO:1, comprising the amino acid sequence set forth in SEQ ID NO:6, and HCDR2 comprising the amino acid sequence of SEQ ID NO:11, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:16, comprising the amino acid sequence set forth in SEQ ID NO:21, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:26, LCDR3 of an amino acid sequence shown in seq id no;

(ii) A heavy chain variable region comprising:

Comprising SEQ ID NO:2, HCDR1 comprising the amino acid sequence of SEQ ID NO:7, and HCDR2 comprising the amino acid sequence of SEQ ID NO:12, HCDR3 of an amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:17, LCDR1 comprising the amino acid sequence of SEQ ID NO:22, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:27, LCDR3 of an amino acid sequence shown in seq id no;

(iii) A heavy chain variable region comprising:

Comprising SEQ ID NO:3, HCDR1 comprising the amino acid sequence of SEQ ID NO:8, and HCDR2 comprising the amino acid sequence of SEQ ID NO:13, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:18, comprising the amino acid sequence set forth in SEQ ID NO:23, and LCDR2 comprising the amino acid sequence of SEQ ID NO:28, LCDR3 of an amino acid sequence shown in seq id no;

(iv) A heavy chain variable region comprising:

Comprising SEQ ID NO:4, HCDR1 comprising the amino acid sequence of SEQ ID NO:9, and an HCDR2 comprising the amino acid sequence of SEQ ID NO:14, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising:

Comprising SEQ ID NO:19, comprising the amino acid sequence set forth in SEQ ID NO:24, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:32, LCDR3 of the amino acid sequence shown in seq id no; or alternatively

(V) A heavy chain variable region comprising:

Comprising SEQ ID NO:5, an HCDR1 comprising the amino acid sequence of SEQ ID NO:10, and an HCDR2 comprising the amino acid sequence of SEQ ID NO:15, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising:

Comprising SEQ ID NO:20, comprising the amino acid sequence set forth in SEQ ID NO:25, and an LCDR2 comprising an amino acid sequence of SEQ ID NO:30, and LCDR3 of the amino acid sequence shown in seq id no.

Embodiment 6. The pharmaceutical combination, method or use according to any one of embodiments 1-5, wherein the anti-CD 47 antibody comprises a heavy chain variable region and a light chain variable region selected from any one of:

(a) The heavy chain variable region comprises a sequence identical to SEQ ID NO:33, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:34, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(b) The heavy chain variable region comprises a sequence identical to SEQ ID NO:35, said light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:36, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(c) The heavy chain variable region comprises a sequence identical to SEQ ID NO:37, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:38, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(d) The heavy chain variable region comprises a sequence identical to SEQ ID NO:39, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:40, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(e) The heavy chain variable region comprises a sequence identical to SEQ ID NO:41, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:42, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(f) The heavy chain variable region comprises a sequence identical to SEQ ID NO:83, said light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:84, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(g) The heavy chain variable region comprises a sequence identical to SEQ ID NO:85, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:86, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no; and

(H) The heavy chain variable region comprises a sequence identical to SEQ ID NO:87, said light chain variable region comprising an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:88, and an amino acid sequence having at least 80% identity thereto.

Embodiment 7. The pharmaceutical combination, method or use according to any one of embodiments 1-6, wherein the anti-CD 47 antibody comprises a heavy chain variable region and a light chain variable region selected from any one of:

(a) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:33, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:34, and a nucleotide sequence shown in seq id no;

(b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:35, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:36, and a nucleotide sequence shown in seq id no;

(c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:37, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:38, and a nucleotide sequence shown in seq id no;

(d) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:39, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:40, and a polypeptide having the amino acid sequence shown in seq id no;

(e) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:41, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:42, and an amino acid sequence shown in seq id no;

(f) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:83, said light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:84, an amino acid sequence shown in seq id no;

(g) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:85, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:86, and a polypeptide having the amino acid sequence shown in seq id no;

(h) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:87, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:88, and a sequence of amino acids shown in seq id no;

(i) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:81, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:82, an amino acid sequence shown in seq id no; or alternatively

(J) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:79, and said light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:80, and an amino acid sequence shown in seq id no.

Embodiment 8. The pharmaceutical combination, method or use according to any one of embodiments 1-7, wherein the anti-CD 47 antibody is selected from any one of the following:

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:89 and a heavy chain according to SEQ ID NO: 91;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:93 and a heavy chain according to SEQ ID NO: 95;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:97 and a heavy chain according to SEQ ID NO:99 light chain;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:101 and a heavy chain according to SEQ ID NO: 103;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:105 and a heavy chain according to SEQ ID NO: 107;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:109 and a heavy chain according to SEQ ID NO: 111;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:113 and a heavy chain according to SEQ ID NO: 115;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:117 and a heavy chain according to SEQ ID NO:119, a light chain;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:121 and a heavy chain according to SEQ ID NO:123 light chain;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:125 and a heavy chain according to SEQ ID NO: 127.

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:129 and a heavy chain according to SEQ ID NO: 131;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:133 and a heavy chain according to SEQ ID NO: 135;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:137 and a heavy chain according to SEQ ID NO:139 light chain;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:141 and a heavy chain according to SEQ ID NO: 143;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:145 and a heavy chain according to SEQ ID NO:147 light chain;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:149 and the heavy chain according to SEQ ID NO: 151;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:153 and a heavy chain according to SEQ ID NO: 155;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:157 and a heavy chain according to SEQ ID NO:159 light chain

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:161 and a heavy chain according to SEQ ID NO:163 light chain;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:165 and the heavy chain according to SEQ ID NO: 167.

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:169 and a heavy chain according to SEQ ID NO: 171;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:173 and a heavy chain according to SEQ ID NO: 175;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:177 and a heavy chain according to SEQ ID NO: 179;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:181 and a heavy chain according to SEQ ID NO:183 light chain; or alternatively

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:185 and a heavy chain according to SEQ ID NO: 187.

Embodiment 9. The pharmaceutical combination, method or use according to any one of embodiments 1-4, wherein the anti-CD 47 antibody comprises:

a heavy chain variable region comprising:

A light chain variable region comprising:

Embodiment 10. The pharmaceutical combination, method or use according to any one of embodiments 1-4, 9, wherein the anti-CD 47 antibody comprises a heavy chain variable region and a light chain variable region selected from any one of:

(a) The heavy chain variable region comprises a sequence identical to SEQ ID NO:87, said light chain variable region comprising an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:88, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(b) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:87, said light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:88, and a sequence of amino acids shown in seq id no;

(c) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:81, and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:82, an amino acid sequence shown in seq id no; or alternatively

(D) The heavy chain variable region comprises the amino acid sequence of SEQ ID NO:79, and said light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:80, and an amino acid sequence shown in seq id no.

Embodiment 11. The pharmaceutical combination, method or use according to any one of embodiments 1-4, 9-10, wherein the anti-CD 47 antibody comprises a polypeptide according to SEQ ID NO:181 and a heavy chain according to SEQ ID NO:183, or the anti-CD 47 antibody comprises a light chain according to SEQ ID NO:185 and a heavy chain according to SEQ ID NO: 187.

Embodiment 12. The pharmaceutical combination, use or method according to any of embodiments 1-11, wherein the anti-CD 47 antibody is of the IgG1, igG2, igG3 or IgG4 isotype.

Embodiment 13. The pharmaceutical combination, use or method according to any of embodiments 1-12, wherein the cancer is primary, recurrent or metastatic.

Embodiment 14. The pharmaceutical combination, use or method according to any of embodiments 1-13, wherein the cancer is a solid tumor, e.g. colorectal cancer.

Embodiment 15. The pharmaceutical combination, use or method according to any of embodiments 1-14, wherein the colorectal cancer is colorectal adenocarcinoma, preferably the colorectal adenocarcinoma is sieve-like acne adenocarcinoma, medullary carcinoma, micro-papillary carcinoma, mucous adenocarcinoma, serrated adenocarcinoma or ring cell carcinoma.

Embodiment 16. The pharmaceutical combination, use or method according to any of embodiments 1-15, wherein the colorectal cancer is colorectal cancer comprising one or more of RAS wild-type, RAS mutant, BRAF wild-type, BRAF mutant, PIK3CA wild-type, pMMR (complete mismatch repair function), MSS (microsatellite stabilization), dMMR (defect in mismatch repair function), MSI (microsatellite instability).

Embodiment 17. The pharmaceutical combination, use or method according to any of embodiments 1-16, wherein the therapeutic dose of the anti-CD 47 antibody is 60mg-3000mg.

Embodiment 18. The pharmaceutical combination, use or method according to any one of embodiments 1-17, wherein the therapeutic dose of the anti-CD 47 antibody is administered every 3-21 days.

Embodiment 19. The pharmaceutical combination, use or method according to any one of embodiments 1-18, wherein a sub-therapeutic dose of said anti-CD 47 antibody is administered prior to the administration of a therapeutic dose of said anti-CD 47 antibody.

Embodiment 20. The pharmaceutical combination, use or method according to any one of embodiments 1-19, wherein the therapeutic dose is achieved by administering the anti-CD 47 antibody in ascending doses.

Embodiment 21. The pharmaceutical combination, use or method according to any of embodiments 1-20, wherein the anti-CD 47 antibody is administered intravenously or/and the tyrosine kinase inhibitor is administered orally.

Embodiment 22. The pharmaceutical combination, use or method according to any of embodiments 1-21, wherein the tyrosine kinase inhibitor is administered in a dose of 6mg to 20mg.

Embodiment 23. The pharmaceutical combination, use or method according to any one of embodiments 1 to 22, wherein the anti-CD 47 antibody and the tyrosine kinase inhibitor are each in the form of a pharmaceutical composition.

Embodiment 24. The pharmaceutical combination, use or method according to any one of embodiments 1-23, wherein the anti-CD 47 antibody and the tyrosine kinase inhibitor are administered simultaneously, sequentially or at intervals.

Embodiment 25. The pharmaceutical combination according to any of embodiments 3-24, wherein the pharmaceutical combination is a non-fixed combination.

Embodiment 26. The pharmaceutical combination according to any one of embodiments 3 to 25, wherein the tyrosine kinase inhibitor is present in the form of a pharmaceutical composition having at least one single dose of 6mg to 20 mg.

Embodiment 27. A kit comprising the pharmaceutical combination of any of embodiments 3-26.

Detailed Description

Example 1: production of anti-CD 47 monoclonal antibodies

Immunization of mice with CD47 protein and acquisition of antibody variable region sequences:

To generate antibodies against CD47, balb/c mice were immunized subcutaneously three times every 2 weeks for 6 weeks using an equal volume of Freund's complete adjuvant (primary immunization) or Freund's incomplete adjuvant (booster immunization) emulsified recombinant hCD47-His Tag protein (ACRObiosystems, catalog number: CD 7-H5227) (50. Mu.g/mouse Balb/c) or hCD47-mFc (ACRObiosystems, catalog number: CD7-H52A 5). A fourth immunization was performed using hCD47-mFc (ACRObiosystems, cat# CD7-H52A 5) protein, 20 μg/mouse, intramuscular injection (aqueous adjuvant). Three days prior to cell fusion, mice were boosted by intravenous injection of antigen without adjuvant. Spleen cells (1X 10 ⁸) from immunized mice were fused with SP2/0 myeloma cells (1.5X10 ⁷) using polyethylene glycol 1500 (Polyethylene Glycol; roche, cat. No.: 10783641001).

Hybridoma cells were selected and cloned. The cloned hybridoma cells were kept and used for cDNA extraction to obtain antibody variable region sequences.

The heavy chain variable region (VH) and light chain variable region (VL) sequences of five murine monoclonal antibodies (2B 2, 2H8, 3F10, 16E5, 14A9 antibodies) were amplified and sequenced from the corresponding hybridoma clones (2B 2, 2H8, 3F10, 16E5, 14 A9) with the following sequence information:

construction and expression of chimeric antibodies:

Chimeric light chains were constructed by chemically synthesizing the genes of the variable regions of the mouse antibodies, respectively linking the VL region of each mouse antibody to the human kappa chain constant region, and linking the mouse VH region to the human IgG4 (EU numbering S228P) constant region. The chimeric light chain expression plasmid and chimeric heavy chain expression plasmid were constructed separately by conventional genetic engineering means, CHO cells (200 mL system, at 10 ⁶ cells/mL) were transfected with 100 μg of each chimeric heavy chain expression plasmid and chimeric light chain expression plasmid and cultured for 6 days. The chimeric antibody in the supernatant was then purified using a protein a column.

Five chimeric antibodies were constructed and expressed, the full length sequences of the chimeric heavy and chimeric light chains and their encoding nucleic acids were as follows: xi2H8 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 89-90; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 91-92), xi2B2 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 97-98; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 99-100), xi3F10 ((chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 101-102; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 103-104)), xi16E5 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 93-94; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 95-96), xi14A9 (chimeric heavy chain and its encoding nucleic acid: SEQ ID NOs: 105-106; chimeric light chain and its encoding nucleic acid: SEQ ID NOs: 107-108).

Antibody humanization design:

The human murine chimeric antibodies of 3F10, 14A9 and 16E5 were humanized using CDR grafting methods (see, e.g., U.S. Pat. No. 5,225,539).

The sequences of the humanized 3F10, 14A9 and 16E5 antibodies and their encoding nucleic acids are as follows: hz3F10-1.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.109-110; light chain and its encoding nucleic acid: SEQ ID NOs.111-112), hz3F10-2.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.113-114; light chain and its encoding nucleic acid: SEQ ID NOs.115-116), hz3F10-3.1 (heavy chain and its encoding nucleic acid: SEQ ID NOs.117-118); light chain and nucleic acid encoding the same: SEQ ID NOs.119 to 120), hz3F10-4.1 (heavy chain and nucleic acid encoding same: SEQ ID NOs.121-122; light chain and nucleic acid encoding the same: SEQ ID NOs.123-124), hz3F10-5.1 (heavy chain and nucleic acid encoding same: SEQ ID NOs.125-126; light chain and nucleic acid encoding the same: SEQ ID NOs.127-128), hz3F10-6.1 (heavy chain and nucleic acid encoding same: SEQ ID NOs.129-130; light chain and nucleic acid encoding the same: SEQ ID NOs.131-132), hz3F10-1.2 (heavy chain and nucleic acid encoding same: SEQ ID NOs.133-134; light chain and nucleic acid encoding the same: SEQ ID NOs.135-136), hz3F10-2.2 (heavy chain and nucleic acid encoding the same: SEQ ID NOs.137-138; light chain and nucleic acid encoding the same: SEQ ID NOs.139 to 140), hz3F10-3.2 (heavy chain and nucleic acid encoding the same: SEQ ID NOs.141-142; light chain and nucleic acid encoding the same: SEQ ID NOs.143 to 144), hz3F10-4.2 (heavy chain and nucleic acid encoding same: SEQ ID NOs.145-146; light chain and nucleic acid encoding the same: SEQ ID NOs.147-148), hz3F10-5.2 (heavy chain and nucleic acid encoding the same: SEQ ID NOs.149-150; light chain and nucleic acid encoding the same: SEQ ID NOs.151-152), hz3F10-6.2 (heavy chain and nucleic acid encoding same: SEQ ID NOs.153-154; light chain and nucleic acid encoding the same: SEQ ID NOs.155-156), hz16E5-1.1 (heavy chain and nucleic acid encoding same: SEQ ID NOs.157-158; light chain and nucleic acid encoding the same: SEQ ID NOs.159-160), hz16E5-1.3 (heavy chain and nucleic acid encoding same: SEQ ID NOs.161-162; light chain and nucleic acid encoding the same: SEQ ID NOs.163-164), hz16E5-1.2 (heavy chain and nucleic acid encoding same: SEQ ID NOs.165-166; light chain and nucleic acid encoding the same: SEQ ID NOs.167 to 168), hz16E5-3.2 (heavy chain and nucleic acid encoding the same: SEQ ID NOs.169-170; light chain and nucleic acid encoding the same: SEQ ID NOs.171-172), hz16E5-3.1 (heavy chain and nucleic acid encoding same: SEQ ID NOs.173-174; light chain and nucleic acid encoding the same: SEQ ID NOs.175 to 176), hz16E5-3.3 (heavy chain and nucleic acid encoding same: SEQ ID NOs.177-178; light chain and nucleic acid encoding the same: SEQ ID NOs.179-180), hz14A9-2.3 (heavy chain and nucleic acid encoding same: SEQ ID NOs.181-182; light chain and nucleic acid encoding the same: SEQ ID NOs.183-184), hz14A9-2.4 (heavy chain and nucleic acid encoding same: SEQ ID NOs.185-186; light chain and nucleic acid encoding the same: SEQ ID NOs.187-188).

Construction and expression of humanized 3F10, 14A9 and 16E5 antibodies:

DNA encoding the full length light and heavy chains of the humanized 3F10, 14A9 and 16E5 antibodies were synthesized and cloned into expression vectors (including, for example, pcdna3.1 vectors disclosed in CN107001463a, pcho1.0 vectors disclosed in CN109422811a, etc.). CHO cells (200 ml system, at 10 ⁶ cells/ml) were transfected with 100 μg of each humanized antibody heavy chain expression plasmid and light chain expression plasmid and incubated with ExpiCHO medium (Gibco; cat# a 29100-01) for 6 days at 37 ℃ in an incubator with 5% co ₂. After centrifugation to obtain a supernatant, the humanized antibody in the supernatant was then purified using a protein a column.

Example 2: biacore-based affinity analysis of anti-CD 47 antibodies

The binding kinetics between Human CD47Protein (Human CD47Protein, his Tag; ACRObiosystems company, catalog number: CD 7-H5227) and Cynomolgus monkey CD47Protein (Cynomolgus/Rhesus macaque CD Protein, his Tag; ACRObiosystems company, catalog number: CD7-C52H 1) and anti-CD 47 antibody (chimeric or humanized) were measured by Biacore, respectively, to obtain an equilibrium dissociation constant K _D (unit M); the Biacore analysis was performed at 25 ℃ and recorded at a data collection rate of 1 Hz.

Polyclonal rabbit anti-mouse IgG (GE, BR-1008-38) was diluted with 10mm sodium acetate pH 5.0 and immobilized onto a reference flow cell and experimental flow cell of a CM5 biosensor chip using an amine coupling kit (GE, BR 10050) to about 15000R μm. At the beginning of each cycle, diluted test antibody (1.5 μg/mL) was injected on the experimental flow cell for 1 minute to be captured. These data indicate that anti-CD 47 antibodies (chimeric and humanized) bind CD47 as measured by Biacore.

Table 1: binding affinity of anti-CD 47 chimeric antibodies to human CD47 and cynomolgus monkey CD47

Table 2: binding affinity of anti-CD 47 humanized antibodies to human CD47 and cynomolgus monkey CD47

Example 3: anti-CD 47 antibodies are based on assays that promote macrophage phagocytosis

The ability of anti-CD 47 antibodies to promote phagocytosis of tumor cells by macrophages containing sirpa on the cell surface was determined based on flow cytometry.

Monocytes were isolated from human peripheral blood mononuclear cells (PBMC, chimaphil, cat# 190056) using a monocyte isolation kit (STEMCELL, cat# 19058), plated in 24-well plates (Greiner bio-one, cat# 662-160), 5X 10 ⁵ cells per well, and induced in an environment of 100ng/mL of stimulating factor M-CSF (R & D Systems, cat# 216-MC-010) for 7 days to induce macrophages. 1X 10 ⁶ target cells HL60 (Shanghai cell institute, catalog number: TCHu) were suspended in 5. Mu. Mol/L CFSE (BD company, catalog number: 565082) fluorescent dye (diluted to the required concentration with PBS buffer), incubated for 15min in a 37℃water bath in the absence of light, and washed with PBS buffer to prepare CFSE-labeled HL60 cells; CFSE-labeled HL60 cells were incubated with 10. Mu.g/mL, 1. Mu.g/mL anti-CD 47 antibodies (chimeric antibodies see Table 3, humanized antibodies see Table 4) at 37℃in a 5% CO ₂ incubator for 1-2h, respectively. The HL60 cells after three incubations were washed with PBS buffer, added to macrophages, incubated at 37℃in a 5% CO ₂ incubator for 2-4h, washed three times with PBS buffer, and then with 260. Mu.l of PBS, 20. Mu.l of a mixture of Anti-APC-CD 14 (APC Mouse Anti-Human CD14, BD Co., catalog number: 555399) and 20. Mu.l of PE-CD11b (PE Mouse Anti-Human CD11b/Mac-1, BD Co., catalog number: 555388) antibody per well, incubated at 4℃in the absence of light for 30min, washed three times with PBS buffer, and digested with pancreatin (Gibco, catalog number: 12604-013) to examine the phagocytic rate (%) of macrophages with a flow cytometer (BD Co., model specification: C6).

The effects of anti-CD 47 chimeric antibodies and anti-CD 47 humanized antibodies on phagocytosis by macrophages are shown in tables 3 and 4, wherein the chimeric antibodies Xi3F10, xi16E5, xi14A9 and anti-CD 47 humanized antibodies have good pro-phagocytosis, and humanized antibodies hz14A9-2.3 and hz14A9-2.4 are more excellent.

Table 3: anti-CD 47 chimeric antibodies are based on assays (phagocytosis rate,%)

Antibody name	10μg/mL	1μg/mL
Xi2B2	4.1	7.3
Xi2H8	3.5	7.1
Xi3F10	59.8	65.7
Xi16E5	28.8	11.5
Xi14A9	44.67	54.17

Table 4: anti-CD 47 humanized antibodies are based on assays (phagocytosis rate,%)

Antibody name	10μg/mL	1μg/mL	Antibody name	10μg/mL	1μg/mL
hz3F10-1.1	43.10	41.25	hz3F10-6.2	44.19	49.32
hz3F10-2.1	55.23	47.24	hz16E5-1.1	4.02	14.79
hz3F10-3.1	58.72	50.60	hz16E5-3.1	24.19	6.37
hz3F10-4.1	58.26	50.26	hz16E5-1.2	17.83	5.1
hz3F10-5.1	58.46	50.45	hz16E5-3.2	2.03	3.02
hz3F10-6.1	60.91	47.57	hz16E5-1.3	4.33	3.03
hz3F10-1.2	38.86	55.52	hz16E5-3.3	4.7	11.62
hz3F10-2.2	34.72	44.19	hz14A9-2.3	74.93	71.48
hz3F10-3.2	37.09	47.04	hz14A9-2.4	81.66	81.65
hz3F10-4.2	31.67	46.24	Hu5F9	72.35	67.5
hz3F10-5.2	29.6	47.06

Example 4: thermal stability of anti-CD 47 antibodies based on nano DSF detection

The high-throughput protein stability analyzer Prometheus NT.48 is a device for obtaining various data such as protein molecular structure stability, aggregation stability, colloidal dispersion stability and the like. The melting temperature (Tm) and the polymerization temperature (Tagg) of the anti-CD 47 chimeric antibody and the anti-CD 47 humanized antibody were measured using the apparatus, and the results are shown in tables 5 and 6, which indicate that the anti-CD 47 antibody has good thermal stability.

Table 5: thermal stability of anti-CD 47 chimeric antibodies

Antibody name	Tm(℃)	Tagg(℃)
Xi2B2	62.99	61.34
Xi2H8	69.77	68.57
Xi3F10	70.51	70.59
Xi16E5	69.89	73.39
Xi14A9	66.73	75.75

Table 6: thermal stability of anti-CD 47 humanized antibodies

Antibody name	Tm(℃)	Tagg(℃)	Antibody name	Tm(℃)	Tagg(℃)
hz3F10-1.1	68.44	72.68	hz14A9-2.4	68.3	68.3
hz3F10-2.1	68.44	72.68	hz3F10-4.2	68.48	61.13
hz3F10-3.1	68.15	61.53	hz3F10-5.2	68.69	62.67
hz3F10-4.1	68.35	62.54	hz3F10-6.2	68.64	61.91
hz3F10-5.1	68.05	62.12	hz16E5-1.1	68.21	61.87
hz3F10-6.1	68.6	68.6	hz16E5-3.1	69.20	61.84
hz3F10-1.2	68.71	61.92	hz16E5-1.2	69.54	61.90
hz3F10-2.2	68.92	62.40	hz16E5-3.2	69.10	61.44
hz3F10-3.2	68.30	61.80	hz16E5-1.3	68.90	73.77
hz14A9-2.3	68.10	73.42	hz16E5-3.3	68.72	73.81

Example 5 pharmacodynamic Activity of anti-CD 47 antibodies in a lymphoma Raji cell mouse blood tumor model

A mouse hematological tumor model is established by using the hematological tumor Raji cells, and an anti-CD 47 antibody is applied to the model to evaluate the pharmacodynamic activity of the antibody.

Raji cell suspension in PBS buffer was 5X 10 ⁶/mL, 0.1 mL/mouse was injected into female NOD/SCID mice by tail vein. After 7 days, the cells were randomly divided into 4 groups of 10 cells each, wherein group 1: igG4 group, group 2: hu5F9 group, group 3: hz3F10-6.1, group 4: hz14A9-2.3 is administrated by intraperitoneal injection, the dosage is 10mg/kg, and the administration is continued for 21 days. The animal life cycle was observed.

As shown in FIG. 1, in the Raji hematological tumor model, the anti-CD 47 antibodies hz14A9-2.3 and hz3F10-6.1 have anti-tumor activity. Survival data for mice showed: the whole group animals died on day 17 of IgG4 group with a median survival of 13.5 days; the animals of the whole group die on day 30 of the Hu5F9 group, and the median survival time is 18.5 days; animals of the whole group of hz14A9-2.3 groups died on day 34 with a median survival of 28.5 days; the animals of the whole group of hz3F10-6.1 group died on day 21 with a median survival of 17.5 days. These data indicate that antibody hz14A9-2.3 has substantially the same efficacy in extending the survival of animals as the other groups, antibodies hz3F10-6.1 and Hu5F 9.

Example 6 toxicity detection of anti-CD 47 antibodies in a single administration

Since CD47 is expressed on erythrocytes and plays a role in the clearance of aged erythrocytes, the toxic effects of anti-CD 47 antibodies in B-hCD47 mice were compared.

B-hCD47 mice were randomly divided into 3 groups: hu5F9 group 3, hz14A9-2.3 group 4, hz3F10-6.1 group 3. The B-hCD47 mice were given a dose of Hu5F9 antibody, hz14A9-2.3 antibody or hz3F10-6.1 antibody at 10mg/kg by single tail vein injection, and changes in red blood cell count (RBC), hemoglobin (HGB) amount, and body weight were measured 2 days, 4 days, 7 days, 10 days, and 14 days after administration.

Table 7: rate of change in body weight of mice after administration (vs before administration)

Days after administration	Hu5F9 10mpk	hz14A9-2.3 10mpk	hz3F10-6.1 10mpk
0	0.0％	0.0％	0.0％
2	-7.0％	2.3％	-13.0％
4	-3.2％	6.3％	-15.8％
7	-5.1％	2.0％	-2.5％
10	4.8％	5.9％	-8.4％
14	5.6％	8.6％	8.2％

Table 8: rate of change of RBC in mice after administration (pre-vs administration)

Days after administration	Hu5F9 10mpk	hz14A9-2.3 10mpk	hz3F10-6.1 10mpk
0	0.0％	0.0％	0.0％
2	-52.8％	-13.6％	-56.7％
4	-61.8％	-16.7％	-75.6％
7	-22.2％	-9.8％	-38.7％
10	-21.6％	-8.5％	-6.6％
14	-7.4％	-2.2％	-2.0％

Table 9: rate of change of HGB in mice after administration (vs before administration)

Days after administration	Hu5F9 10mpk	hz14A9-2.3 10mpk	hz3F10-6.1 10mpk
0	0.0％	0.0％	0.0％
2	-52.7％	-13.3％	-57.1％
4	-57.8％	-18.5％	-74.7％
7	-16.5％	-9.9％	-39.2％
10	-12.8％	-10.0％	-6.0％
14	-1.2％	-3.9％	-3.0％

As shown in Table 7, FIGS. 2 and 3, only mice in the hz14A9-2.3 group had a sustained increase in body weight, and mice in the Hu5F9 and hz3F10-6.1 groups had a decrease in body weight and an increase in body weight after administration; the hz3F10-6.1 group was reduced by a higher magnitude than the Hu5F9 group. As shown in tables 8-9, figures 4-7, the RBCs and HGBs of the three groups of animals all fell after dosing, reaching a minimum 4 days after dosing, and then began to rise. RBC and HGB substantially return to normal 7 days after dosing in the hz14A9-2.3 group and substantially return to normal in the other two groups at least 10 days after dosing. The descending amplitude of RBC and HGB after 4 days of administration is as follows from high to low: hz3F10-6.1 > Hu5F9 > hz14A9-2.3. It was found that antibody hz14A9-2.3 had no significant toxicity, which was superior to the positive antibody Hu5F9.

EXAMPLE 7 efficacy of hz14A9-2.3, an Luoti Nib hydrochloride alone or in combination with DiFi mice with human colorectal cancer

The experimental animals were NOD-SCID mice, 5 weeks old, available from Fukang Biotechnology Co., ltd. Production license number: SCXK (Beijing) 2019-0008; animal pass number 110322211100393952. Feeding environment: SPF stage. Human colorectal cancer DiFi cells were given from the Ming Kangde Biotechnology Co. DiFi cells were cultured at 37℃in an air incubator with 5% CO ₂. Passaging for 2 times a week, digesting the cells with pancreatin when the cells are in exponential growth phase, collecting the cells, counting, and inoculating. Each mouse was inoculated subcutaneously with 8 x 10 ⁶ diffi mouse cells and the tumors were grown to 100-150mm ³, grouped, and given Intravenous (IV) medication 2 times per week (BIW) for a total of 4 times; or intragastric administration (i.g.), 1 time per day (QD), 21 times total, administration volume 0.1mL/10g body weight; the doses and dosing schedules are shown in Table 10.

The experimental index is to examine the influence of the drug on the tumor growth, and the specific index is T/C% or tumor growth inhibition rate (TGI) and tumor inhibition rate.

Tumor diameter was measured twice weekly with vernier calipers and tumor volume (V) was calculated as:

V=1/2×a×b ², where a and b represent length and width, respectively.

T/C (%) = (T-T ₀)/(C-C ₀) ×100, where T, C is tumor volume at the end of the test and control group animal experiments, respectively; t ₀、C ₀ is the tumor volume at the beginning of the test and control group animal experiments, respectively.

Tumor growth inhibition rate (TGI%) =100-T/C (%).

Tumor growth inhibition rate (TGI%) =100- (T-T ₀)/T ₀ ×100 when tumor regression occurs

Tumor Partial Regression (PR) is defined if the tumor is reduced from the starting volume, i.e., T < T ₀ or C < C ₀; if the tumor completely disappeared, it is defined as complete tumor regression (CR).

At the end of the experiment (D20), the animals were sacrificed under CO ₂ anesthesia, then dissected for tumor removal and photographed.

TABLE 10 efficacy of hz14A9-2.3, an Luoti Nib hydrochloride alone or in combination on DiFi mice with human colorectal cancer

Note that: randomly grouping, wherein the first administration time is D0; BIW: 2 times per week; IV: intravenous injection; i.g.: gastric lavage administration; QD: 1 time per day.

The results are shown in Table 10, FIGS. 8-9, the anti-CD 47 antibody hz14A9-2.3 (1.5 mg/kg, IV, BIW X2) has an inhibitory effect on the growth of human colorectal cancer DiFi mice subcutaneous transplantable tumors, with a tumor inhibition rate of 55%; an Luoti Ni (1 mg/kg, i.g., QD×21) had a tumor inhibition of 74% for DiFi mice transplanted subcutaneously; the tumor inhibition rate of the hz14A9-2.3 (1.5 mg/kg, IV, BIW multiplied by 2) and An Luoti Ni (1 mg/kg, i.g., QD multiplied by 21) hydrochloride combined use is improved to 96 percent, which is obviously stronger than the single drug curative effect (P < 0.01); the mice in the hIgG4 group have the maximum weight reduction of 10.3 percent (D20), which indicates that the tumor model has injury effect on the mice; the maximum weight loss of mice in the Hz14A9-2.3 and An Luoti Nigroup of hydrochloric acid was 2.2% (D20) and 5.1% (D20), respectively, and the maximum weight loss of mice in the Hz14A9-2.3+ An Luoti Nigroup was 3.1% (D20), indicating that the above drugs can improve the general status of mice, possibly by inhibiting tumor growth in mice.

Example 8 phase I clinical trial of anti-CD 47 antibodies alone or in combination in patients with advanced malignancy

The main purpose is as follows: safety and tolerability of anti-CD 47 antibodies alone (stage Ia) or in combination (stage Ib) in patients with advanced malignancy was assessed, and Dose Limiting Toxicity (DLT), maximum Tolerated Dose (MTD) (if any) or Optimal Biologic Dose (OBD), and phase II recommended dose (RP 2D) were determined.

The secondary purpose is as follows: assessing the Pharmacokinetic (PK) profile of the anti-CD 47 antibody in patients with advanced malignancy when the anti-CD 47 antibody is used alone or in combination; assessing the initial effectiveness of the anti-CD 47 antibody alone or in combination to treat advanced malignancy; assessing the immunogenicity of the anti-CD 47 antibody; the Pharmacodynamic (PD) profile of anti-CD 47 antibodies, alone or in combination, was evaluated.

Mainly comprises the following steps:

Age 1.18 years ∈75 years (calculated as date of informed consent was signed); the male and female are not limited; ECOG scores 0-1 score; survival T3 months was expected.

2. Malignant solid tumors need to satisfy: a. malignant solid tumors are explicitly diagnosed by histology or cytology. b. Disease progression/recurrence consistent with RECIST1.1 definition. c. Standard treatment failed or no standard treatment method.

Test design and dosing regimen:

The study used an open dose escalation trial design with two phases of single dose escalation of Ia and combined dose escalation of Ib.

The Ia single dose escalation phase, standard-compliant patients were assigned sequentially to 5 anti-CD 47 antibody dose groups (60 mg, 180mg, 600mg, 1200mg and 1800 mg) in order of entry. Each patient in the group received the target dose of anti-CD 47 antibody treatment, once weekly intravenous infusion was tentatively given, for a treatment period of no more than 2 years for every 4 weeks (28 days).

A combination dose escalation stage of Ib, selecting the MTD or OBD or RP2D dose or dosing regimen determined in the single dose escalation stage of Ia, and performing dose escalation of the combination in different indication queues. Wherein, the queue of the anti-CD 47 antibody and An Luoti Ni hydrochloride combined treatment is incorporated into the patients with recurrent/metastatic advanced solid tumor, and the An Luoti Ni capsule adopted by An Luoti Ni (10/12 mg, QD, d1-14/q3 w) scheme treatment is oral capsule with the specification of 8mg, 10mg and 12mg (calculated by C ₂₃H ₂₂FN ₃O ₃) and is produced and provided by the company of the pharmaceutical industry group Co., inc. of the day.

The inclusion/exclusion criteria, dosing regimen and study procedure of the Ib combination dose escalation stage will be based on the results and experience of the Ia single dose escalation stage and the revision of the regimen in time.

The effectiveness and safety evaluation were performed according to the standards.

The anti-CD 47 antibody used in the study is hz14A9-2.3 injection, which is a sterile injection for intravenous administration and is produced and provided by Nanjing cisxin pharmaceutical Co., ltd. The present study also relates to the evaluation of anti-CD 47 antibodies in combination with other therapeutic agents, not described herein.

Claims

The use of an anti-CD 47 antibody and a tyrosine kinase inhibitor in the manufacture of a medicament for the treatment of cancer in a subject, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof,
A pharmaceutical combination comprising:

(a) anti-CD 47 antibodies, and

(B) A tyrosine kinase inhibitor, wherein the tyrosine kinase inhibitor is a compound of formula I or a pharmaceutically acceptable salt thereof,
Pharmaceutical combination or use according to any one of claims 1-2, wherein the pharmaceutically acceptable salt of the compound of formula I is the hydrochloride, preferably the dihydrochloride, of 1- [ [ [4- (4-fluoro-2-methyl-1H-indol-5-yl) oxy-6-methoxyquinolin-7-yl ] oxy ] methyl ] cyclopropylamine.
The pharmaceutical combination or use of any one of claims 1-3, wherein the anti-CD 47 antibody comprises:

(i) A heavy chain variable region comprising:

Comprising SEQ ID NO:5, an HCDR1 comprising the amino acid sequence of SEQ ID NO:10, and an HCDR2 comprising the amino acid sequence of SEQ ID NO:15, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising:

Comprising SEQ ID NO:20, comprising the amino acid sequence set forth in SEQ ID NO:25, and an LCDR2 comprising an amino acid sequence of SEQ ID NO:30, LCDR3 of the amino acid sequence shown in seq id no;

(ii) A heavy chain variable region comprising:

Comprising SEQ ID NO:1, comprising the amino acid sequence set forth in SEQ ID NO:6, and HCDR2 comprising the amino acid sequence of SEQ ID NO:11, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:16, comprising the amino acid sequence set forth in SEQ ID NO:21, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:26, LCDR3 of an amino acid sequence shown in seq id no;

(iii) A heavy chain variable region comprising:

Comprising SEQ ID NO:2, HCDR1 comprising the amino acid sequence of SEQ ID NO:7, and HCDR2 comprising the amino acid sequence of SEQ ID NO:12, HCDR3 of an amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:17, LCDR1 comprising the amino acid sequence of SEQ ID NO:22, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:27, LCDR3 of an amino acid sequence shown in seq id no;

(iv) A heavy chain variable region comprising:

Comprising SEQ ID NO:3, HCDR1 comprising the amino acid sequence of SEQ ID NO:8, and HCDR2 comprising the amino acid sequence of SEQ ID NO:13, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising: comprising SEQ ID NO:18, comprising the amino acid sequence set forth in SEQ ID NO:23, and LCDR2 comprising the amino acid sequence of SEQ ID NO:28, LCDR3 of an amino acid sequence shown in seq id no; or alternatively

(V) A heavy chain variable region comprising:

Comprising SEQ ID NO:4, HCDR1 comprising the amino acid sequence of SEQ ID NO:9, and an HCDR2 comprising the amino acid sequence of SEQ ID NO:14, HCDR3 of the amino acid sequence shown in seq id no; and

A light chain variable region comprising:

Comprising SEQ ID NO:19, comprising the amino acid sequence set forth in SEQ ID NO:24, and an LCDR2 comprising the amino acid sequence of SEQ ID NO:32, and LCDR3 of the amino acid sequence shown in seq id no.
The pharmaceutical combination or use of any one of claims 1-4, wherein the anti-CD 47 antibody comprises a heavy chain variable region and a light chain variable region selected from any one of:

(a) The heavy chain variable region comprises a sequence identical to SEQ ID NO:87, said light chain variable region comprising an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:88, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(b) The heavy chain variable region comprises a sequence identical to SEQ ID NO:33, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:34, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(c) The heavy chain variable region comprises a sequence identical to SEQ ID NO:35, said light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:36, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(d) The heavy chain variable region comprises a sequence identical to SEQ ID NO:37, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:38, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(e) The heavy chain variable region comprises a sequence identical to SEQ ID NO:39, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:40, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(f) The heavy chain variable region comprises a sequence identical to SEQ ID NO:41, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:42, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no;

(g) The heavy chain variable region comprises a sequence identical to SEQ ID NO:83, said light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:84, an amino acid sequence having at least 80% identity to the sequence set forth in seq id no; and

(H) The heavy chain variable region comprises a sequence identical to SEQ ID NO:85, and the light chain variable region comprises an amino acid sequence having at least 80% identity to the sequence set forth in SEQ ID NO:86, and an amino acid sequence having at least 80% identity.
The pharmaceutical combination or use according to any one of claims 1-5, wherein the anti-CD 47 antibody is selected from any one of the following:

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:181 and a heavy chain according to SEQ ID NO:183 light chain;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:89 and a heavy chain according to SEQ ID NO: 91;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:93 and a heavy chain according to SEQ ID NO: 95;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:97 and a heavy chain according to SEQ ID NO:99 light chain;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:101 and a heavy chain according to SEQ ID NO: 103;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:105 and a heavy chain according to SEQ ID NO: 107;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:109 and a heavy chain according to SEQ ID NO: 111;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:113 and a heavy chain according to SEQ ID NO: 115;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:117 and a heavy chain according to SEQ ID NO:119, a light chain;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:121 and a heavy chain according to SEQ ID NO:123 light chain;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:125 and a heavy chain according to SEQ ID NO: 127.

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:129 and a heavy chain according to SEQ ID NO: 131;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:133 and a heavy chain according to SEQ ID NO: 135;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:137 and a heavy chain according to SEQ ID NO:139 light chain;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:141 and a heavy chain according to SEQ ID NO: 143;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:145 and a heavy chain according to SEQ ID NO:147 light chain;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:149 and the heavy chain according to SEQ ID NO: 151;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:153 and a heavy chain according to SEQ ID NO: 155;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:157 and a heavy chain according to SEQ ID NO:159 light chain

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:161 and a heavy chain according to SEQ ID NO:163 light chain;

the anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:165 and the heavy chain according to SEQ ID NO: 167.

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:169 and a heavy chain according to SEQ ID NO: 171;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:173 and a heavy chain according to SEQ ID NO: 175;

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:177 and a heavy chain according to SEQ ID NO: 179;

Or alternatively

The anti-CD 47 antibody comprises an amino acid sequence according to SEQ ID NO:185 and a heavy chain according to SEQ ID NO: 187.
The pharmaceutical combination or use according to any one of claims 1-6, wherein the anti-CD 47 antibody is of the IgG1, igG2, igG3 or IgG4 isotype.
The pharmaceutical combination or use according to any one of claims 1-7, wherein the cancer is primary, recurrent or metastatic.
The pharmaceutical combination or use according to any one of claims 1-8, wherein the cancer is a solid tumor;

Preferably, the colorectal cancer is colorectal adenocarcinoma, preferably, the colorectal adenocarcinoma is sieve-like acne adenocarcinoma, medullary carcinoma, micro-papillary carcinoma, mucous adenocarcinoma, serrated adenocarcinoma or ring cell carcinoma;

Preferably, the colorectal cancer is colorectal cancer comprising one or more of RAS wild-type, RAS mutant, BRAF wild-type, BRAF mutant, PIK3CA wild-type, pMMR (complete mismatch repair function), MSS (microsatellite stabilization), dMMR (defect in mismatch repair function), MSI (microsatellite instability).
The pharmaceutical combination, use or method according to any one of claims 1-9, wherein the therapeutic dose of the anti-CD 47 antibody is 60mg-3000mg.
The pharmaceutical combination, use or method according to any one of claims 1-10, wherein the anti-CD 47 antibody is administered intravenously or/and the tyrosine kinase inhibitor is administered orally.
The pharmaceutical combination, use or method according to any one of claims 1-11, wherein the tyrosine kinase inhibitor is administered in a dose of 6mg-20mg.
The pharmaceutical combination according to any one of claims 2-12, wherein the pharmaceutical combination is a non-fixed combination.
The pharmaceutical combination according to any one of claims 2-13, wherein the tyrosine kinase inhibitor is present in the form of a pharmaceutical composition having at least one single dose of 6mg-20 mg.
A kit comprising the pharmaceutical combination of any one of claims 2-14.